CN103202228A - One-step seedling and efficient in-vitro propagation method with gynura bicolor leaves - Google Patents
One-step seedling and efficient in-vitro propagation method with gynura bicolor leaves Download PDFInfo
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Abstract
The invention relates to a one-step seedling and efficient in-vitro propagation method for Begoniaceae gynura bicolor, which is an endangered plant with homology of medicine and food. The method provided by the invention comprises the following steps of: preparing a culture medium, and adjusting the culture medium through preparing, subpackaging and sterilizing; carrying out sterile inoculating directly on gynura bicolor aseptic seedling leaves which are taken as explants; culturing the inoculated culture medium in a culture chamber for 70-80 days under appropriate conditions, wherein the number of a single explant induced seedlings is 20 above; and decapping, transplanting regenerating plants to appropriate conditions, and culturing without hardening-seedling, wherein the commodity seedling survival rate achieves 100%. According to the method provided by the invention, the gynura bicolor tissue culture seedling regeneration period is short; the original seed property is kept effectively; the vegetative propagation coefficient is improved obviously; the operation is simple and convenient; the culture program is simple; the seedling cost is lowered obviously; and the method can act as an effective technology of industrialized production of high-quality seedlings of Begoniaceae gynura bicolor.
Description
Technical field
The invention belongs to biological technical field, relate to the method for tissue culture of rare or endangered species rapid seedling cultivation, be specifically related to the forming seedling through one step culture method for quickly breeding that the leaf tissue of medicinal and edible plant Begoniaceae Gynura bicolor is cultivated.
Background technology
Gynura bicolor (
Begonia fimbristipulataHance) be Begoniaceae Begonia meat herbaceous plant, the peculiar species of China, call red leaf, fimbriatestipulate begonia herb, diffusing blood, being common in longly under cheuch area, steep cliff crack of stone or the sparse woods of height above sea level 200 ~ 1 120 m has on the moist rock of liver moss, liking warm wet cold air waits, generally be the population of clustering or growing thickly and distribute, accompanying plant has liver moss, small-sized fern, shrub etc.Gynura bicolor all has distribution on the ground such as Shenzhen, Dongguan, Zhaoqing and Jiangling in Guangdong, but the most famous with the Gynura bicolor of Dinghu Hill, Zhaoqing, its type specimen namely picks up from the Dinghu Hill, Guangdong.Though Gynura bicolor distributes wide, be limited in the special habitat, cliff place, the mountain valley growth of curling up with sea of clouds fog, smoke, mists and clouds is best, and population is discreteness and distributes, and population quantity very easily is subjected to the influence of environmental change.
The Gynura bicolor leaf is rich in anthocyanin, and its sour-puckery flavor, cool in nature both can be used as medicine, the drink that also can keep healthy, and tool is clearing heat and detoxicating, slake thirst and help produce saliva, activating blood circulation and reducing swelling, the effect of moistening dryness and relieve cough, and complete stool has higher ornamental value and medicinal health value.Yet, because its dangerously steep suitable environment, lower biological yield, add that long-term destructiveness plucks, cause wild resource exhausted day by day, now by " rare plant of China " book be evaluated as endangered species low danger grade (Lower Risk, LR).
Gynura bicolor seminal propagation germination rate is extremely low, by bulb carry out the division propagation coefficient lower, be difficult to satisfy the demand in market.Utilizing tissue culture technique is the effective way of accelerating the Gynura bicolor large-scale breeding.It is explant that existing document is got its aseptic seedling blade usually, carry out the cell induction of embryoid, or inducing and be differentiated to form indefinite bud again by callus, or differentiate the bud of growing thickly by Gynura bicolor in vitro tissues such as indefinite bud, bulb bud or bulb or organ, and then be transferred to the root media root induction, form complete plant at last.Regeneration plant (being commonly referred to a bottle seedling) still needs finally to form the commodity seedling through just transplanting after the hardening program.As seen, the fast breeding technique of Gynura bicolor is still continued to use traditional training method of organizing so far, a plurality of stages such as, subculture cultivation foster through just being commissioned to train, culture of rootage and acclimatization and transplants, program is loaded down with trivial details, and it is consuming time to take a lot of work, and exists growing-seedling period long, germplasm easily produces variation, the more high shortcoming of cost.Therefore, be necessary to study that it is easy, forming seedling through one step culture technology efficiently.The forming seedling through one step culture method be exactly explant after dedifferentiation, do not need to transfer on the differential medium, but directly differentiation again on former medium, bud behind the normally first root of the order of differentiation is until forming healthy and strong full stand.It is short that it has the regeneration period, the regeneration frequency height, and easy to operate, the cultivation program is simple, does not influence the rate of increase, keeps former specific character and is convenient to desirable features such as large-scale production.
Summary of the invention
The object of the invention is to provide a kind of endangered plants Gynura bicolor forming seedling through one step culture quick propagating technology, reaches the effect that explant induction namely obtains the shiploads of merchandise seedling.It can significantly be simplified the program of growing seedlings, shortens growing-seedling period, improves vegetative propagation coefficient, reduce variation frequency and easy and simple to handle, is the effective way that realizes that the batch production of Gynura bicolor high quality seedling is produced.Simultaneously, the propagation that can be similar plant provides technical basis with the integrated breeding of taking root.
A kind of Gynura bicolor blade forming seedling through one step culture efficient in vifro culture method of the present invention may further comprise the steps: (1) medium is prepared, and comprising: preparation, packing, sterilization; Described culture medium preparation is: adding 6-benzyl aminopurine in every liter of MS minimal medium is that 6-BA 0.25 ~ 0.5 mg, α-Nai Yisuan are that NAA 0.4 ~ 0.8 mg, indolebutyric acid are IBA 0.2 mg, carragheen 7 g, sucrose 30 ~ 40 g, and the pH that mixes the back medium is adjusted to 6.0; The packing of described medium is: the medium after will preparing is heated to agar and dissolves fully, is sub-packed in equably while hot in the clean blake bottle; The sterilization of described medium is: the blake bottle that will add medium sterilization 15 minutes under 121 ℃, 105 Kpa, and the cooling of sterilization back is standby; (2) explant is handled and inoculation, comprising: adopting Gynura bicolor aseptic seedling blade is explant, directly carries out aseptic inoculation, namely under aseptic condition, the tissue culture sterile seedling placed on the aseptic workbench cut blade, with blade inoculation on ready medium, the sterile sealing bottleneck; (3) cultivate, comprising: will connect the blake bottle immigration culturing room of planting and carry out inducing and cultivating of seedling, and form healthy and strong Gynura bicolor regrowth clump, and be used for the uncork transplanting; (4) regeneration plant is transplanted: without hardening, the direct uncork of regeneration plant that step (3) obtains is transplanted to suitable condition cultivation.
According to the further feature of method of the present invention, in the described step (1), described medium is: add 6-BA 0.25 ~ 0.5 mg, NAA 0.4 ~ 0.8 mg, IBA 0.2 mg, carragheen 7g, sucrose 30 ~ 40g in every liter of MS minimal medium.
According to the further feature of method of the present invention, in the described step (2), the employing leaf directly is that the Gynura bicolor aseptic seedling blade of 0.8 ~ 1.5 cm is explant, directly carries out aseptic inoculation.
According to the further feature of method of the present invention, in the described step (3), the condition of culture of seedling is: temperature is 25 ± 1 ℃, and intensity of illumination is 8 ~ 10 μ mol.m
-2.s
-1, light application time is 10 hours every days, incubation time is 70 ~ 80 days.The diligent inspection between culture period found to pollute seedling and in time removed.
According to the further feature of method of the present invention, in the described step (3), before described seedling clump uncork is transplanted, with 15 μ mol.m
-2.s
-1Intensity of illumination added intense light irradiation 10 days.
According to the further feature of method of the present invention, in the described step (4), the tissue culturing seedling of clustering directly or be separated into the Cong Miao form that has 3 ~ 5 plant and transplant.
According to the further feature of method of the present invention, in the described step (4), transplanting medium adopts complete peat soil, regulates the pH buffering range of matrix between 4.0 ~ 4.5.
According to the further feature of method of the present invention, in the described step (4), transplanting medium adopts peat soil: perlite is 3:1(
V/
V) mixed-matrix, regulate the pH buffering range of matrix between 4.0 ~ 4.5.
According to the further feature of method of the present invention, in the described step (4), the condition of culture after the transplanting is: day temperature is 25 ℃, and evening, temperature was 22 ℃, just 25 ℃/22 ℃ (light/dark); Relative moisture is 90%, intensity of illumination 15-18 μ mol.m
-2.s
-1, light application time is 12 hours every days.
According to the further feature of method of the present invention, in the described step (4), the culture fluid after the transplanting is running water or 1/4 MS culture fluid.
Experiment shows that above-mentioned transplanting meets the characteristic that wild Gynura bicolor population is grown thickly, and seedling early growth is very fast, and the survival rate of seedling reaches 100% after 35 days, and growing way is healthy and strong, thereby the artificial Gynura bicolor of cultivating better keeps the original seed feature.
The present invention has following advantage and effect:
1, the experiment proved that, in the method for the invention, adopt MS+0.25 ~ 0.5 mg/L 6-BA+0.4 ~ 0.8 mg/L NAA+0.2 mg/L IBA+30 ~ 40 g/L sucrose+7 g/L carragheens as the cultivation of inducing of Gynura bicolor forming seedling through one step culture, explant induction reaches 100% for the incidence of clump bud, average number of seedling 25 ~ 30 strains of single explant, tissue culturing seedling's well-grown keeps former specific character substantially, the quality tunnel.
2, the experiment proved that, in the method for the invention, adopt Gynura bicolor aseptic seedling blade as explant, it is zero that the survival rate of explant reaches the 100%(pollution rate), inductivity is more than 93.7%, and a plurality of indefinite bud points can directly be induced and differentiate to each explant.
3, the experiment proved that, in the method for the invention, in the preferred condition of culture of explant, with intensity of illumination 10 μ molm
-2S
-1, incubation time is 70 ~ 80 d, growth quality situation the best of Cong Miao, and 3 ~ 5 of the individual plant numbers of sheets, about 1.7 ~ 3.5 mm of bulb diameter, seedling clump root length is 2.0 ~ 3.8 cm, height of seedling 2.5 ~ 3.3 cm.Before tissue culturing seedling's uncork was transplanted, appropriateness added intense light irradiation (about 15 μ molm
-2S
-1) about 10 days, seedling is grown thickly long more healthy and strong.
4, the experiment proved that, in the method for the invention, without hardening, the tissue culturing seedling of clustering can directly or disperse to transplant, and the survival rate of transplanting reaches more than 90%, wherein, the tissue culturing seedling that to cluster is separated into the clump bud form that has 3 ~ 5 bulbs, be transplanted to [peat soil: perlite=3:1 (
V/
V)] mixed-matrix in cultivate, the survival rate of 35 days metaplexus seedlings reaches 100%, and growing way is healthy and strong.
5, compare with existing propagation technique, technical method of the present invention has extremely significantly been simplified the Gynura bicolor program of growing seedlings, shorten growing-seedling period, improve vegetative propagation coefficient, keep former specific character, the cultivation program is simple, reaches the effect that explant induction namely obtains the shiploads of merchandise seedling, is the effective way that realizes that the batch production of endangered plants Gynura bicolor high quality seedling is produced.
6, the method for the invention need not task equipment, overcomes the influence that the germ contamination of explant causes growing seedlings, and is easy and simple to handle.
In sum, the present invention is material with the Gynura bicolor blade, study different growth regulator combination formulas to the influence of Gynura bicolor forming seedling through one step culture, by screening and culturing base component, optimize condition of culture, simplify the tissue culture technique flow process, explore without the tradition tissue and cultivate alternately the continue forming seedling through one step culture quick-breeding method of program of too many levels, set up the efficient of medicinal and edible plant Begoniaceae Gynura bicolor rapid propagation in vitro in imminent danger, easy, stable practical technique system, reach once inoculation and then cultivate into healthy and strong full stand, and significantly improve vegetative propagation coefficient, can directly apply to the batch production production of Gynura bicolor commodity seedling.
Embodiment
Material and reagent
Material: the Gynura bicolor aseptic seedling derives from Nature Reserve Tianxi section, Dinghu Hill, Guangdong Province, is genuine wild provenance, gets its aseptic blade as explant after supporting through just being commissioned to train.
Reagent: 6-BA(6-benzyl aminoadenine, purity 〉=98%, moisture≤0.5%), the NAA(methyl, purity 99.9%), the IBA(indolebutyric acid, purity 99.9%) and other reagent (analyzing pure AR) of preparation MS medium, all buy from last seamount Pu chemical industry Co., Ltd.
Instrument and condition of culture
The YXQ-LS-75SII type vertical pressure steam sterilizer that Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd. produces is for the usefulness of medium sterilization; The single single face clean work station of SW-CJ-1FD type that Purifying Equipment Co., Ltd., Suzhou produces is for the usefulness of material switching.All place temperature (25 ± 1) ℃, relative moisture 65 ~ 70%, intensity of illumination 8 ~ 10 μ molm after the switching
-2S
-1Culturing room in cultivate light application time h every days 10.
Embodiment 1:
(1) culture medium preparation: be minimal medium with MS, every liter is added 6-BA 0.25 mg, NAA 0.4 mg and IBA 0.2 mg, carragheen 7 g, sucrose 30 g, and the pH that mixes the back medium is adjusted to 6.0; Every bottle of about 30 mL of packing place sterilising conditions to be made as: sterilization is 15 minutes in 121 ℃, the high-pressure sterilizing pot of 105 Kpa, takes out blake bottle, cools off standby;
(2) explant is handled and inoculation: vaccination ways routinely, cutting the Gynura bicolor aseptic seedling blade that leaf directly is about 0.8 ~ 1.5 cm is explant, blade directly is inoculated in the above-mentioned medium (MS+0.25 mg/L 6-BA+0.4 mg/L NAA+0.2 mg/L IBA+30 g/L sucrose+7 g/L carragheens), 2 explants of every bottle graft kind, the sterile sealing bottleneck;
(3) cultivate: postvaccinal blake bottle is moved into culturing room cultivate, condition of culture is: 25 ± 1 ℃ of temperature, relative air humidity 65 ~ 70%, intensity of illumination 8 ~ 10 μ molm
-2S
-1, light application time 10 h/ days.Cultivate after 15 days, the survival rate 100% of explant, blade expands, and leaf margin upwarps, and faint yellow transparent healing cell (or tissue) occurs, inductivity 93%.Leaf margin callus cells of superficial layer or blade surface cell differentiate intensive bud point then, and bud point increases gradually and forms green orbicule, and leaf bud structure, differentiation rate are arranged is 100%.Cultivate after 35 days, part bud point forms the intensive indefinite bud state of growing thickly, and the explant blade changes block bud clump, highly about 0 ~ 6 mm of bud clump, bud clump differentiation rate 100% into.Cultivate after 50 days, Miao Cong begins to form, and base portion has pale fish-egg shape bulb to form, and leaf is tiny, and color is yellowish green.Cultivate after 65 days, Miao Cong basically forms, yellow green, and bulb manifests substantially and increases, and part is unconverted organizes yellow or withered for the bud clump of seedling, and planting percent is 75%.Cultivated 80 days, the aseptic seedling that can obtain to cluster, the regeneration plant morphosis is complete.Uncork final singling this moment, each explant induction seedling number be greater than 20 strains, seedling clump average height 2.33 cm, bulb diameter range 1.7 ~ 3.0 mm, rooting rate 100%, long 2.0 ~ 4.0 cm of root, seedling clump root system FAQ.
(4) tissue culturing seedling transplants: the bottle seedling adds intense light irradiation (about 15 μ molm through appropriateness
-2S
-1) cultivated about 10 days, without hardening, the Gynura bicolor tissue culturing seedling that will cluster takes out, and flowing water directly is transplanted to peat soil 3 after cleaning and removing medium: perlite 1 (
V/V) mixed-matrix in, placing temperature is 25 ℃/22 ℃ (light/dark), relative moisture is 90%, intensity of illumination 15-18 μ mol.m
-2.s
-1Growth conditions under manually cultivate, with the pouring of running water or 1/4MS culture fluid.Tissue culturing seedling's survival rate is 90% after 35 days, and growth better.
Embodiment 2:
Other operation is with embodiment 1, and difference is: (in (1), the prescription of medium is (MS+0.25 mg/L 6-BA+0.6 mg/L NAA+0.2 mg/L IBA+30 g/L sucrose+7 g/L carragheens) to step; Step (in (3), cultivate after 15 days, the survival rate 100% of explant, blade obviously expands, and leaf margin upwarps, the inductivity 100% of healing cell (or tissue).Cultivate after 35 days, the explant blade changes block bud clump into, and differentiation rate is 100%, and highly about 0 ~ 7 mm of bud clump, the bud long speed of growing thickly is very fast, and has a small amount of intact leaf to form.Simultaneously, the bud clump organizes the inboard to send white suede hair, and suede hair ramp subsequently is the radicula of the white fine hair of close quilt, and root tissue is abundanter.Cultivate after 45 days, Miao Cong begins to form, and base portion has bulb to manifest, tiny, the expansion of blade, peak green.Cultivate after 65 days, Miao Cong forms, and growing way is better, and part is unconverted organizes yellow or withered for the bud clump of seedling, and planting percent is 85%.Cultivated about 75 days, and can obtain the aseptic seedling that stalwartness is clustered, the regeneration plant morphosis is complete.Uncork final singling this moment, each explant induction seedling number be greater than 25 strains, seedling clump average height 2.63 cm, and bulb diameter range 2.0 ~ 3.0 mm, there is bronzing cell tier covering on the surface, rooting rate 100%, long 3.0 ~ 4.0 cm of root, seedling clump root system is intensive, quality is better.Step (in (4), without hardening, cultivation matrix is peat soil 3: perlite 1 (
V/V) mixed-matrix, after 35 days growth of seedling in order, survival rate 100%.
Embodiment 3:
Other operation is with embodiment 2, and difference is: (in (1), the prescription of medium is (MS+0.25 mg/L 6-BA+0.8 mg/L NAA+0.2 mg/L IBA+30 g/L sucrose+7 g/L carragheens) to step; Step (in (3), cultivate after 15 days, the survival rate 100% of explant, blade significantly expands and arches upward, and leaf margin upwarps, callus induction rate 100%.Cultivate after 35 days, the explant blade changes block bud clump rapidly into, and differentiation rate is 100%, and highly about 2 ~ 7 mm of bud clump, bud are grown thickly long vigorous, and has a large amount of intact leafs to form.Simultaneously, bud clump base portion has a large amount of white fine hair shape radiculas to generate, and root system is abundant.Cultivate after 40 days, Miao Cong begins to form, and the base portion bulb is obvious, vane extension, green.Cultivate after 65 days, Miao Cong forms, and growing way is vigorous, and Cong Miao highly surpasses 3.5 cm, and planting percent is 100%.Cultivated about 70 days, and can obtain the aseptic seedling that stalwartness is clustered, the complete stalwartness of regeneration plant morphosis.Uncork final singling this moment, each explant induction seedling number be greater than 30 strains, seedling clump average height 3.55 cm, and bulb diameter range 2.0 ~ 3.5 mm, there is bronzing cell tier covering on the surface, rooting rate 100%, long 2.5 ~ 3.5 cm of root, the intensive Cheng Cong of root system, optimal quality.Step (in (4), seedling percent 100% after 35 days, the seedling condition is healthy and strong.
Embodiment 4:
Other operation is with embodiment 1, and difference is: (in (1), the prescription of medium is (MS+ 0.5 mg/L 6-BA+0.4 mg/L NAA+0.2 mg/L IBA+30 g/L sucrose+7 g/L carragheens) to step; Step (in (3), cultivate after 15 days, the survival rate 100% of explant, blade expands, and leaf margin upwarps, and faint yellow transparent healing cell (or tissue) occurs, inductivity 100%.Cultivate after 35 days, the explant blade changes block bud clump into, and differentiation rate is 100%, and highly about 0 ~ 3.5 mm of bud clump, the bud long speed of growing thickly is slower.Cultivate after 65 days, Miao Cong begins to form, and short and small, major part be the tender leaf of gauffer, and a small amount of mounted blade is the not exclusively ripe seedling clump of bright yellow, and planting percent is 45%, and part is unconverted to be organized yellow for the bud clump of seedling and also wither.At this moment, Miao Cong organizes the inboard to send white fine hair shape radicula, and adventive root is more.Cultivated about 80 days, the aseptic seedling that can obtain to cluster, the regeneration plant morphosis is complete substantially.Uncork final singling this moment, each explant induction seedling number be greater than 12 strains, seedling clump average height 1.58 cm, and bulb is less, diameter range 1.2 ~ 2.5 mm, rooting rate 90%, long 1.2 ~ 3.0 cm of root, seedling clump root system is sparse.(in (4), tissue culturing seedling's survival rate is 80% to step after 35 days, and growth is normal.
Embodiment 5:
Other operation is with embodiment 4, and difference is: (in (1), the prescription of medium is (MS+ 0.5 mg/L 6-BA+0.6 mg/L NAA+0.2 mg/L IBA+30 g/L sucrose+7 g/L carragheens) to step; Step (in (3), is cultivated after 15 days the survival rate 100% of explant, inductivity 100%.Cultivate after 35 days, the explant blade is divided into block bud clump, differentiation rate 100%, and highly about 0 ~ 4.0 mm of bud clump, the bud long speed of growing thickly is slower.Cultivate after 65 days, Miao Cong begins to form, short and small a little less than, major part be the tender leaf of gauffer, a small amount of mounted blade is the not exclusively ripe seedling clump of bright yellow, planting percent is 57%, part is unconverted to be organized yellow for the bud clump of seedling and also withers.At this moment, Miao Cong organizes the inboard only to send a small amount of white fine hair shape radicula.Cultivated about 80 days, the aseptic seedling that can obtain to cluster, the regeneration plant morphosis is complete substantially.Uncork final singling this moment, each explant induction seedling number be greater than 18 strains, seedling clump average height 1.56 cm, and bulb is less, diameter range 1.2 ~ 3.0 mm, rooting rate 60%, long 1.2 ~ 2.2 cm of root, seedling clump radical is less.(in (4), tissue culturing seedling's survival rate is 60% to step after 35 days, and it is normal substantially to grow.
Embodiment 6:
Other operation is with embodiment 1 or example 2, and difference is: (in (1), medium sucrose is 40 g/L to step; Step (in (3), is cultivated after 15 days the survival rate 100% of explant, inductivity 100%.Cultivate after 30 days, bud clump differentiation rate is 100%, about 0 ~ 6.0 mm of clump bud height, and growth rate is very fast.Cultivate after 45 days, Miao Cong begins to form, and base portion has bulb to manifest, and there are obvious white fleck, leaf back pale red in mounted blade, surface.Cultivate after 65 days, Miao Cong forms, and growing way is better, and planting percent is more than 90%.Cultivated about 75 days, and can obtain the aseptic seedling that stalwartness is clustered, the regeneration plant morphosis is complete, and the original seed feature is obvious.Step (in (4), without hardening, cultivation matrix is complete peat soil or peat soil 3: perlite 1 (
V/V) mixed-matrix all can, after 35 days seedling grow fine genuine, survival rate 100%.
Embodiment 7:
Other operation is with embodiment 3, and difference is: (in (1), the prescription of medium is (MS+0.25 mg/L 6-BA+0.8 mg/L NAA+0.2 mg/L IBA+40 g/L sucrose+7 g/L carragheens) to step; Step (in (3), is cultivated after 15 days the survival rate 100% of explant, callus induction rate 100%.Cultivate after 30 days bud clump differentiation rate 100%, highly about 3 ~ 7 mm of bud clump.Simultaneously, bud clump base portion has a large amount of white fine hair shape radiculas to generate, and root system is abundant.Cultivate after 40 days, Miao Cong begins to form, and the base portion bulb is obvious.Cultivate after 65 days, seedling clump growing way is vigorous, highly surpasses 4.0 cm, vane extension, and there is obvious white fleck on the surface, the leaf back pale red, there is bronzing cell tier covering on the bulb surface, and planting percent is 100%.Cultivated about 70 days, and can obtain the aseptic seedling that stalwartness is clustered, the original seed feature is obvious.Uncork final singling this moment, each explant induction seedling number be greater than 30 strains, seedling clump average height 3.8 cm, and bulb diameter range 2.5 ~ 3.7 mm, rooting rate 100%, long 2.7 ~ 3.5 cm of root, root system is abundant.Step (in (4), without hardening, cultivation matrix is complete peat soil or peat soil 3: perlite 1 (
V/V) mixed-matrix all can, seedling is good after 35 days, keeps former specific character substantially, quality is genuine.
Embodiment 8:
Other operation is with embodiment 4 or example 5, and difference is: (in (1), medium sucrose is 40 g/L to step; Step (in (3), is cultivated after 15 days the survival rate 100% of explant, inductivity 95%.Cultivate after 35 days, the explant blade is divided into block bud clump, differentiation rate 100%, and highly about 0 ~ 4.0 mm of bud clump, the bud long speed of growing thickly is slower.Cultivate after 65 days, Miao Cong begins to form, and short and small, major part is the tender leaf of gauffer, a small amount of mounted blade, and surperficial adularescent fleck, planting percent are 50%.Cultivated about 80 days, the light red colour cell training seedling that can obtain to cluster, the regeneration plant morphosis is complete substantially.Step (in (4), cultivate in complete peat soil or peat soil 3: perlite 1 (
V/V) mixed-matrix, tissue culturing seedling's survival rate is 70% after 35 days, growth is normal.
Embodiment 9:
Other operation is with embodiment 1,2,3,4 and 5, and difference is: (in (4), the bottle seedling adds intense light irradiation (about 15 μ molm through appropriateness to step
-2S
-1) cultivated about 10 days, without hardening, the Gynura bicolor tissue culturing seedling that will cluster takes out, flowing water directly is transplanted in the complete peat soil matrix after cleaning and removing medium, and placing temperature is 25 ℃/22 ℃ (light/dark), relative moisture is 90%, intensity of illumination 15-18 μ mol.m
-2.s
-1Growth conditions under manually cultivate, usually with the pouring of running water or 1/4MS culture fluid.Tissue culturing seedling's survival rate is more than 80 ~ 100% after 35 days, and growth is normal.
Claims (10)
1. a Gynura bicolor blade forming seedling through one step culture efficient in vifro culture method is characterized in that, may further comprise the steps:
(1) medium is prepared, and comprising: preparation, packing, sterilization;
Described culture medium preparation is: adding 6-benzyl aminopurine in every liter of MS minimal medium is that 6-BA 0.25 ~ 0.5 mg, α-Nai Yisuan are that NAA 0.4 ~ 0.8 mg, indolebutyric acid are IBA 0.2 mg, carragheen 7 g, sucrose 30 ~ 40 g, and the pH that mixes the back medium is adjusted to 6.0;
The packing of described medium is: the medium after will preparing is heated to agar and dissolves fully, is sub-packed in equably while hot in the clean blake bottle;
The sterilization of described medium is: the blake bottle that will add medium sterilization 15 minutes under 121 ℃, 105 Kpa, and the cooling of sterilization back is standby;
(2) explant is handled and inoculation, comprising: adopting Gynura bicolor aseptic seedling blade is explant, directly carries out aseptic inoculation, namely under aseptic condition, the tissue culture sterile seedling placed on the aseptic workbench cut blade, with blade inoculation on ready medium, the sterile sealing bottleneck;
(3) cultivate, comprising: will connect the blake bottle immigration culturing room of planting and carry out inducing and cultivating of seedling, and form healthy and strong Gynura bicolor regrowth clump, and be used for the uncork transplanting;
(4) regeneration plant is transplanted: without hardening, the direct uncork of regeneration plant that step (3) obtains is transplanted to suitable condition cultivation.
2. method according to claim 1 is characterized in that, in the described step (1), described medium is: add 6-BA 0.25 ~ 0.5 mg, NAA 0.4 ~ 0.8 mg, IBA 0.2 mg, carragheen 7g, sucrose 30 ~ 40g in every liter of MS minimal medium.
3. method according to claim 1 is characterized in that: in the described step (2), adopting leaf directly is that the Gynura bicolor aseptic seedling blade of 0.8 ~ 1.5 cm is explant, directly carries out aseptic inoculation.
4. method according to claim 1 is characterized in that, in the described step (3), the condition of culture of seedling is: temperature is 25 ± 1 ℃, and intensity of illumination is 8 ~ 10 μ mol.m
-2.s
-1, light application time is 10 hours every days, incubation time is 70 ~ 80 days.
5. method according to claim 1 is characterized in that: in the described step (3), before described seedling clump uncork is transplanted, with 15 μ mol.m
-2.s
-1Intensity of illumination added intense light irradiation 10 days.
6. method according to claim 1 is characterized in that: in the described step (4), the tissue culturing seedling of clustering directly or be separated into the Cong Miao form that has 3 ~ 5 plant and transplant.
7. method according to claim 1, it is characterized in that: in the described step (4), transplanting medium adopts complete peat soil, regulates the pH buffering range of matrix between 4.0 ~ 4.5.
8. method according to claim 1 is characterized in that: in the described step (4), transplanting medium adopts peat soil: perlite is 3:1(
V/
V) mixed-matrix, regulate the pH buffering range of matrix between 4.0 ~ 4.5.
9. method according to claim 1, it is characterized in that: in the described step (4), the condition of culture after the transplanting is: day temperature is 25 ℃, and evening, temperature was 22 ℃, and relative moisture is 90%, intensity of illumination 15-18 μ mol.m
-2.s
-1, light application time is 12 hours every days.
10. method according to claim 1 is characterized in that, in the described step (4), the culture fluid after the transplanting is running water or 1/4 MS culture fluid.
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| CN107646682A (en) * | 2017-10-15 | 2018-02-02 | 陈金水 | A kind of nakedcaule groundsel tissue culture and rapid propagation method |
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